Isolation and/or preservation of dendritic cells for prostate cancer immunotherapy

ABSTRACT

Methods and compositions for use of human dendritic cells to activate T cells for immunotherapeutic responses against primary and metastatic prostate cancer are disclosed. In one embodiment, human dendritic cells, after exposure to a prostate cancer antigen or specific antigenic peptide, are administered to a prostate cancer patient to activate the relevant T cell responses in vivo. In an alternate embodiment, human dendritic cells are exposed to a prostate cancer antigen or specific antigenic peptide in vitro and incubated or cultured with primed or unprimed T cells to activate the relevant T cell responses in vitro. The activated T cells are then administered to a prostate cancer patient. Methods and compositions for human dendritic cells with extended life span and cryopreserved dendritic cells are disclosed.

1. FIELD OF THE INVENTION

The present invention relates to compositions and methods of isolatingand/or preserving and using human dendritic cells for immunotherapy forcancer. In particular, the invention relates to methods for the use ofdendritic cells for the activation and expansion of large numbers ofprostate antigen specific T cells for use in adoptive cellularimmunotherapy against prostate cancer, as well as methods for the use ofprostate antigen pulsed dendritic cells as vaccines and/orimmunotherapeutics to slow or inhibit the growth of primary ormetastatic prostate cancer.

2. BACKGROUND OF THE INVENTION 2.1. Prostate Cancer

Prostate cancer is the second leading cause of death from cancer amongmen. In fact, prostate cancer is the most common (noncutaneous) cancerdiagnosed in the American male and is steadily increasing, as a resultof the increasing population of older men as well as greater awarenessand earlier diagnosis of the disease. See, Wright et al., 1995, Urol.Oncol. 1:18-28. In 1995, it is projected that over 244,000 men will bediagnosed with prostate cancer in this year. There will be 40,400deaths. The life time risk for men, to suffer prostate cancer is now 1in 8 for Caucasians, and an estimated 1 in 7 for African Americans. Highrisk groups are those with a positive family history or AfricanAmericans. Over a lifetime, above 25% of the men diagnosed with prostatecancer will die of the disease. Wingo et al., 1995, CA Cancer J. Clin.45(2):8-30. Moreover, many patients who do not die of prostate cancerrequire treatment to ameliorate symptoms such as pain, bleeding, andurinary obstruction. Thus, prostate cancer is also a major cause ofsuffering and of health care expenditures. Catalona, W. J., 1994, NewEng. J. Med. 331:996-1004.

Cytotoxic chemotherapy is largely ineffective in treating prostatecancer. A combination of agents is no more effective than a singleagent, and the addition of chemotherapy to hormonal therapy does notimprove survival. Eisenberger, M. A., 1988, Chemotherapy for prostatecarcinoma. In: Wittes, R. E., ed. Consensus Development Conference onthe Management of Clinically Localized Prostate Cancer. NCI monographsNo. 7 Washington D.C.: Government Printing Office: 151-153 (NIHpublication no. 88-3005). Accordingly, there is a great demand forimproved prostate cancer therapeutics.

2.2 Cancer Immunotherapy

It is well established that the immune system can function to kill tumorcells, including both primary and metastatic cancer cells. Indeed,evidence that the immune system recognizes the presence of neoplasticcancerous cells is supported by the existence of infiltratinglymphocytes in tumor tissues (Haskill et al., 1978, Contemp. Top.Immunobiol. 8:107-170; Vose and Moore, 1985, Semin. Hematol. 22:27-40).Yet, for reasons that have not been completely clear, despite thepresence of immune cells, tumors often prevail and not only survive butmetastasize to distant sites with unrestricted growth.

Recent advances in the understanding of T cell activation andrecognition of target cells have begun to permit some progress indevelopment of T cell mediated cancer immunotherapy (Schwartz, 1992,Cell 71:1065-1068; Pardoll, 1992, Curr. Opin. Immunol. 4:619-623).

In its most general form, the generation of an immune response beginswith the sensitization of helper (T_(H), CD4⁺) and cytotoxic (CD8⁺) Tcell subsets through their interaction with antigen presenting cells(APC) that express major histocompatibility (MHC)-class I or class IImolecules associated with antigenic fragments (i.e., specific amino acidsequences derived from the antigen which bind to MHC I and MHC II forpresentation on the cell surface). The sensitized or primed CD4⁺ T cellsproduce lymphokines that participate in the activation of B cells aswell as various T cell subsets. The sensitized CD8⁺ T cells increase innumbers in response to lymphokines and are capable of destroying anycells that express the specific antigenic fragments associated withmatching MHC-encoded class I molecules. Thus, in the course of acancerous tumor, CTL eradicate cells expressing cancer associated orcancer specific antigens, thereby limiting the progression of tumorspread and disease development.

Various methods for immunotherapy of a number of particular cancers havebeen suggested; however, to date no one has developed any therapeuticmethod that successfully elicits an effective immunotherapeutic responseagainst primary or metastatic prostate cancer.

2.3 Dendritic Cells for Antigen Presentation

Antigen presenting cells (APC) are particularly important in elicitingan effective immune response.

By definition, APC not only can present antigens to T cells withantigen-specific receptors, but can provide all the signals necessaryfor T cell activation. Such signals are incompletely defined, butprobably involve a variety of cell surface molecules as well ascytokines or growth factors. Further, the factors necessary for theactivation of naive or unprimed T cells may be different from thoserequired for the re-activation of previously primed memory T cells. Theability of APC to both present antigens and deliver signals for T cellactivation is commonly referred to as an accessory cell function.Although monocytes and B cells have been shown to be competent APC,their antigen presenting capacities in vitro appear to be limited to there-activation of previously sensitized T cells. Hence, they are notcapable of directly activating functionally naive or unprimed T cellpopulations.

The term “dendritic cells” refers to a diverse population ofmorphologically similar cell types found in a variety of lymphoid andnon-lymphoid tissues (Steinman, 1991, Ann. Rev. Immunol. 9:271-296).These cells include lymphoid DC of the spleen, Langerhans cells of theepidermis, and veiled cells in the blood circulation. Although they arecollectively classified as a group based on their morphology, highlevels of surface MHC-class II expression, and absence of certain othersurface markers expressed on T cells, B cells, monocytes, and naturalkiller cells, it is presently not known whether they derive from acommon precursor or can all function as APC in the same manner. Itshould be noted that a putative novel antigenic marker of human DCrecognized by monoclonal antibody CMRF-44 has been reported (Hock etal., 1994 Immunol. 83(4):P573-581).

Recent studies have described methods for the isolation and expansion ofhuman DC's, including, from human peripheral blood. [Macatonia et al.,1991, Immunol. 74:399-406; O'Doherty et al., 1993, J. Exp. Med.178:1067-1078 (isolation); and Markowicz et al., 1990, J. Clin. Invest.85:955-961; Romani et al., 1994, J. Exp. Med. 180:83-93; Sallusto etal., 1994, J. Exp. Med. 179:1109-1118; Berhard et al., 1995, Cancer Res.55:1099-1104 (expansion)]. PCT Publication WO 94/02156 describes amethod for isolating human DC's to present antigens to induce antigenspecific T cell-mediated responses. Adoptive cellular immunotherapy anduse of the DC's against infectious diseases and cancer are mentioned.

Citation or identification of any reference in Section 2 (or any othersection) of this application shall not be construed as an admission thatsuch reference is available as prior art to the present invention.

3. SUMMARY OF THE INVENTION

The present invention provides methods, and compositions, for use ofdendritic cells to activate T cells for immunotherapeutic responsesagainst primary or metastatic prostate cancer. The DC's obtained fromhuman donors, after exposure to a prostate cancer antigen or antigenicfragment, are administered to a prostate cancer patient to activate therelevant T cell responses in vivo. Alternatively, the DC's are exposedto a prostate cancer antigen in vitro and incubated with primed orunprimed T cells to activate the relevant T cell responses in vitro. Theactivated T cells are then administered to a prostate cancer patient. Ineither case, the DC's are advantageously used to elicit animmunotherapeutic growth inhibiting response against a primary ormetastatic prostate tumor.

In one embodiment, the invention provides a method for producing acancer growth inhibiting response, which comprises administering, to aprostate cancer patient in need thereof, an effective amount ofactivated T cells, in which the T cells were activated in vitro byexposure to human dendritic cells exposed to a prostate cancer antigen.In another embodiment, the invention provides a method for producing acancer growth inhibiting response, which comprises administering, to aprostate cancer patient in need thereof, an effective amount of humandendritic cells, exposed in vitro to a prostate cancer antigen, suchthat after administration the human dendritic cells elicit an immuneresponse or augment an existing immune response against the prostatecancer.

Prostate cancer antigens useful for the methods and compositions of theinvention include but are not limited to: a lysate of LNCaP cells, amembrane preparation of LNCaP cells, a lysate of prostate tumor cells ofa prostate cancer patient, a membrane preparation of prostate tumorcells of a prostate cancer patient, purified prostate specific membraneantigen (PSMA a/k/a PSM), a peptide having the amino acid sequenceLLHETDSAV (SEQ. ID. NO. 1), a peptide having the amino acid sequenceALFDIESKV (SEQ. ID. NO. 2), a peptide having the amino acid sequenceXL(or M)XXXXXXV(or L) (SEQ. ID. NO. 3) where X represents any aminoacid, purified prostate specific antigen (PSA), and a purified prostatemucin antigen recognized by monoclonal antibody PD41.

The present invention, further provides compositions comprising isolatedhuman dendritic cells exposed to a prostate cancer antigen(s), as wellas cryopreserved isolated human dendritic cells and extended life spanhuman dendritic cells which are useful for eliciting immunotherapeuticresponses against primary and/or metastatic prostate cancer.

4. BRIEF DESCRIPTION OF THE FIGURES

The present invention may be more fully understood by reference to thefollowing detailed description of the invention, examples of specificembodiments of the invention and the appended figures in which:

FIG. 1 represents histograms of results of flow cytometric analyses ofcultured dendritic cells which illustrate the expressed cell surfaceantigens. The cells were cultured in the presence of GM-CSF and IL-4.The (topmost) upper left histogram represents background fluorescencestaining using the secondary (2°) antibody, i.e., goat anti-mouse Ig, inthe absence of any primary (1°) antibody. The rest of the histogramsrepresent fluorescence staining in the presence of 2° antibody and eachof the enumerated 1° antibodies, respectively, anti-CD1a, anti-CD3,anti-CD4, anti-CD11c, anti-CD14, anti-CD19, anti-B7/BB1 and anti-HLA-DRantibodies. See text Section 6 for details.

FIG. 2 graphically illustrates in vitro activation of T cells by tetanustoxoid (TT) presented by autologous DC's. See text Section 6 for detailsof the assays conducted in triplicate. The extent of T cellproliferation, represented by ³HTdR incorporated (cpm), is presentedalong the y axis. Three different culture conditions for the T cells arerepresented along the x axis: tetanus toxoid alone (+TT); dendriticcells alone (+DC); and tetanus toxoid with dendritic cells (+DC-TT).Individual standard deviations are shown.

FIG. 3 graphically illustrates in vitro activation of T cells from fourprostate cancer patients by presentation of prostate cancer antigen byautologous dendritic cells. Data from each individual patient is shownby different bar graph patterns as indicated. The extent of T cellproliferation, represented by ³H-Thymidine eHTdR) incorporated (cpm), ispresented along the y axis. Three different culture conditions for the Tcells are represented along the x axis: a prostate cancer antigen alone(+LNCaP lysate); dendritic cells alone (+DC); and a prostate cancerantigen with dendritic cells (+DC+LNCaP lysate). See text Section 6.4.for details.

FIG. 4 is a histogram illustrating fluorescence flow cytometric analysisof the population of T cells proliferated in response to autologous DCpresentation of LNCaP lysate as antigen. Background fluorescencerepresented by experiments with 2° antibody (goat anti-mouse Ig) aloneis shown by the dotted line histogram. Fluorescence obtained inexperiments using the 2° antibody with the 1° antibody (anti-CD8)antibody are shown by the solid line histogram. See text Section 6 fordetails.

FIG. 5 graphically illustrates cytolytic activity of T cells stimulated,in vitro, in response to autologous DC presentation of LNCaP lysate asantigen. Average percent specific lysis of target cells is represented(y axis) as a function of (x axis) Effector (activated T cells): TargetCells (autologous DC's presenting LNCaP

or autologous DC's alone

). Experiments were performed in triplicate and the values presentedrepresent the average; standard error of the mean for all experimentswas <10%. See text Section 6.4 for details

FIG. 6 illustrates in vitro activation of T cells (PBMC) by presentationof prostate cancer antigen by previously frozen autologous dendriticcells. The prostate cancer antigen used was purified prostate specificmembrane antigen (PSMA). The extent of T cell proliferation, representedby ³H-TdR incorporated is shown along the y axis. Values are expressedas mean cpm±standard deviation (S.D.). Each experiment was conducted in4 replicates. Three different culture conditions for the T cells arerepresented along the x axis: T cells plus dendritic cells (PBMC+DC); Tcells plus PSMA alone (PBMC+PSMA); and T cells plus dendritic cells plusPSMA (PBMC+DC+PSMA). See text Section 7.1 for details.

FIG. 7 illustrates in vitro activation of T cells by presentation ofantigens by previously frozen autologous extended life span dendriticcells. The extent of T cell proliferation, represented by ³H-TdRincorporated is shown along the x axis. Values are expressed as meanvalue±S.D. after subtraction of average background (PBMC+medium only).Five different cultures of T cells are represented along the y axis: Tcells plus extended life span dendritic cells plus tetanus toxoid(+EBV-cells+TT); T cells plus extended life span dendritic cells plusprostate antigen (+EBV-cells+LNCaP); T cells plus extended life spandendritic cells plus no antigen (+EBV-cells); T cells plus tetanustoxoid alone (+TT); and T cells plus prostate antigen alone (+LNCaP).See text Section 8 for details.

FIG. 8 illustrates that a peptide having amino acid sequence LLHETDSAV(comprising a portion of the amino acid sequence of PSMA) can stimulateproliferation of a mixed population of T cells (PBMC) obtained fromperipheral blood of a prostate cancer patient. The extent of T cellproliferation, represented by ³HTdR incorporation (cpm), is presentedalong the y axis. See text Section 9 for details.

FIG. 9 (A-C) is a histogram illustrating fluorescence flow cytometricanalysis of the population of T cells proliferated in response to thepeptide LLHETDSAV as illustrated in FIG. 8. FIG. 9A illustratesbackground fluorescence obtained in experiments using 2° antibody only.FIG. 9B illustrates bound fluorescence obtained using the 2° antibodyand anti-CD4 as the 1° antibody. FIG. 9C illustrates bound fluorescenceobtained using 2° antibody and anti-CD8 as the 1° antibody. See textSection 9 for details.

FIG. 10 illustrates that a peptide having amino acid sequence ALFDIESKVcan stimulate T cells (PBMC) obtained from purified blood of prostatecancer patients. The extent of T cell proliferation, represented by³HTdR incorporation (cpm), is greater in HLA-A2(+) cells. See textSection 9 for details.

FIG. 11 (A-E) present results of a clinical trial demonstrating thatadministration of dendritic cells exposed to a peptide having amino acidsequence LLHETDSAV (PSM-P1) or amino acid sequence ALFDIESKV (PSM-P2)induced an enhanced immune response in late stage prostate cancerpatients as assessed by ratio of T cell proliferation pre- andpost-administration of the DC's.

The ratio is designated “stimulation ratio”. FIG. 11(A) illustratesresults with patients infused with peptide PSM-P1 alone (Group I); FIG.11(B), with patients infused with peptide PSM-P2 alone (Group II); FIG.11(C), with patients infused with autologous dendritic cells alone(Group III); FIG. 11(D), with patients infused with dendritic cells andPSM-P1 (Group IV); and FIG. 11(E), with patients infused with dendriticcells and PSM-P2 (Group V). See text Section 10 for details.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods, and compositions, for use ofdendritic cells to activate T cells for immunotherapeutic responsesagainst primary or metastatic prostate cancer. The DC's obtained fromhuman donors, once exposed to a prostate cancer antigen or specificantigenic peptide, are administered to a prostate cancer patient toactivate the relevant T cell responses in vivo. Alternatively, the DC'sare exposed to a prostate cancer antigen or specific antigenic peptidein vitro and incubated with primed or unprimed T cells to activate therelevant T cell responses in vitro. The activated T cells are thenadministered to a prostate cancer patient. In either case, the DC's areadvantageously used to elicit an immunotherapeutic growth inhibitingresponse against a primary or metastatic prostate tumor.

Solely for ease of explanation, the description of the invention isdivided into the following sections: (1) methods for obtaining orisolating dendritic cells, including DC's with extended lifespan orcryopreserved DC's; (2) prostate specific antigens for presentation byDC's; and (3) applications or methods of use of DC's to stimulate Tcells against prostate cancer in vitro and in vivo.

5.1. Isolation of Dendritic Cells

Human DC's are obtained from any tissue where they reside includingnon-lymphoid tissues such as the epidermis of the skin (Langerhanscells) and lymphoid tissues such as the spleen, bone marrow, lymph nodesand thymus as well as the circulatory system including blood (bloodDC's) and lymph (veiled cells). Human peripheral blood is an easilyaccessible ready source of human DC's and is used as a source accordingto a preferred embodiment of the invention. Cord blood is another sourceof human DC's and in cases where a male is born into a family known tobe at high risk for prostate cancer, cord blood can be used as a sourceof DC's which can be cryopreserved for later use, if needed.

Because DC's occur in low numbers in any tissues in which they reside,including human peripheral blood, DC's must be enriched or isolated foruse. Any of a number of procedures entailing repetitive density gradientseparation, positive selection, negative selection or a combinationthereof are used to obtain enriched populations or isolated DC's.Examples of such methods for isolating DC's from human peripheral bloodinclude: O'Doherty et al, 1993, J. Exp. Med. 178:1067-1078; Young andSteinman, 1990, J. Exp. Med. 171:1315-1332; Freudenthal and Steinman,1990, PNAS USA 87:7698-7702; Macatonia et al., 1989, Immunol.67:285-289; and Markowicz and Engleman, 1990, J. Clin. Invest.85:955-961. A method for isolating DC's from human peripheral bloodwhich avoids exposure of the cells to sheep red blood cells and/or fetalcalf serum is described in PCT Publication WO94/02156. An example of amethod for isolating DC's from lymphoid tissue is described in Macatoniaet al., 1989, J. Exp. Med. 169:1255-1264.

Once the DC's are obtained, they are cultured in appropriate culturemedium to expand the cell population and/or maintain the DC's in a statefor optimal antigen uptake, processing and presentation. Particularlyadvantageous for maintenance of the proper state of “maturity” of DC'sin in vitro culture is the presence of both granulocyte/macrophagecolony stimulating factor (GM-CSF) and interleukin 4 (IL-4). Preferredis a combination of GM-CSF: IL-4 in concentration of about 500 units/mlof each. A recent study reveals optimal antigen presentation by“immature” vs. mature DC (Koch et al., J. Immunol. 155:93-100). ImmatureDC's may be preferred according to certain embodiments of the presentinvention.

As illustrated in the examples, infra, in Section 6, human DC's wereisolated from peripheral blood of prostate cancer patients, and afterabout 7 days in culture, about 20-50 fold higher numbers of DC'scompetent and able to activate prostate antigen specific T cells wererecovered compared to those directly isolated from peripheral blood.

According to a preferred embodiment of the invention, DC's are obtainedfrom a prostate cancer patient to be treated. The DC's are used toactivate autologous T cells of the patient, either in vitro or in vivo,for cancer immunotherapy and/or tumor growth inhibition.

According to an alternate embodiment, DC's are obtained from a healthyindividual known not to be suffering from prostate cancer. The relevantHLA antigens (both class I and II, e.g., HLA-A, B, C and DR) on theindividual's PBMC's are identified and DC's which match the prostatecancer patient, in terms of HLA antigens, are isolated and expanded asdescribed above. For example, in certain instances, a late stageprostate cancer patient who has been treated with radiation and/orchemotherapy agents often are not able to provide sufficient orefficient DC's. Thus, DC's from healthy HLA-matched individuals, such assiblings, can be obtained and expanded using any of the methodsdescribed above and incubated in vitro with a prostate cancer antigen toelicit activated T cells for immunotherapy and/or tumor growthinhibition in the HLA-matched prostate cancer patient.

According to another embodiment of the invention, “extended life spandendritic cells” are used. Human cells have a finite life span in vitrousually limited to approximately 50-70 population doublings beforeundergoing apoptosis. As used herein, the term “extended life spandendritic cells” is intended to mean DC's that have been geneticallymodified so that they can be expanded in in vitro cell culture mediumfor an extended period of time, including but not limited to at leastabout 100 additional population doublings. Extended life span DC's areobtained, for example, by EBV-transformation of DC's obtained fromperipheral blood of prostate cancer patients, or by insertion into DC's,using techniques known to those skilled in the art, of a specific cellcycle regulatory gene including but not limited to a gene which encodescyclin A, B, D or E or retinoblastoma protein.

As illustrated in the examples, infra, in Section 8, extended life spanDC's have been obtained by EBV transformation of a population ofisolated DC's. A number of clones have been obtained which express therelevant DC characteristic antigens and continue to grow and divide wellafter normal isolated DC's do not.

According to yet another embodiment of the invention, DC's can bepreserved, e.g., by cryopreservation either before exposure or followingexposure to a prostate cancer antigen.

Cryopreservation agents which can be used include but are not limited todimethyl sulfoxide (DSO) (Lovelock and Bishop, 1959, Nature183:1394-1395; Ashwood-Smith, 1961, Nature 190:1204-1205), glycerol,polyvinylpyrrolidone (Rinfret, 1960, nn. N.Y. Acad. Sci. 85:576),polyethylene glycol (Sloviter and Ravdin, 1962, Nature 196:548),albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol,D-mannitol (Rowe et al., 1962, Fed. Proc. 21:157), D-sorbitol,i-inositol, D-lactose, choline chloride (Bender et al., 1960, J. Appl.Physiol. 15520), amino acids (Phan The Tran and Bender, 1960, Exp. CellRes. 20:651), methanol, acetamide, glycerol monoacetate (Lovelock, 1954,Biochem. J. 56:265), and inorganic salts (Phan The Tran and Bender,1960, Proc. Soc. Exp. Biol. Med. 104:388; Phan The Tran and Bender,1961, in Radiobiology, Proceedings of the Third Australian Conference onRadiobiology, Ilbery, P. L. T., ed., Butterworth, London, p. 59).

A controlled slow cooling rate is critical. Different cryoprotectiveagents (Rapatz et al., 1968, Cryobiology 5(1):18-25) and different celltypes have different optimal cooling rates (see, e.g., Rowe and Rinfret,1962, Blood 20:636; Rowe, 1966, Cryobiology 3(1):12-18; Lewis et al.,1967, Transfusion 7(1):17-32; and Mazur, 1970, Science 168939-949 foreffects of cooling velocity on survival of marrow-stem cells and ontheir transplantation potential). The heat of fusion phase where waterturns to ice should be minimal. The cooling procedure can be carried outby use of, e.g., a programmable freezing device or a methanol bathprocedure.

Programmable freezing apparatuses allow determination of optimal coolingrates and facilitate standard reproducible cooling. Programmablecontrolled-rate freezers such as Cryomed or Planar permit tuning of thefreezing regimen to the desired cooling rate curve.

After thorough freezing, cells can be rapidly transferred to a long-termcryogenic storage vessel. In a preferred embodiment, samples can becryogenically stored in liquid nitrogen (−196° C.) or its vapor (−165°C.). Such storage is greatly facilitated by the availability of highlyefficient liquid nitrogen refrigerators, which resemble large Thermoscontainers with an extremely low vacuum and internal super insulation,such that heat leakage and nitrogen losses are kept to an absoluteminimum.

Considerations and procedures for the manipulation, cryopreservation,and long term storage of hematopoietic stem cells, particularly frombone marrow or peripheral blood, is largely applicable to the DC's ofthe invention. Such a discussion can be found, for example, in thefollowing references, incorporated by reference herein: Gorin, 1986,Clinics in Haematology 15(1) 19-48; Bone-Marrow Conservation, Cultureand Transplantation, Proceedings of a Panel, Moscow, Jul. 22-26, 1968,International Atomic Energy Agency, Vienna, pp. 107-186.

Other methods of cryopreservation of viable cells, or modificationsthereof, are available and envisioned for use (e.g., cold metal-mirrortechniques; Livesey and Linner, 1987, Nature 327:255; Linner et al.,1986, J. Histochem. Cytochem. 34(9):1123-1135; see also U.S. Pat. No.4,199,022 by Senken et al., U.S. Pat. No. 3,753,357 by Schwartz, U.S.Pat. No. 4,559,298 by Fahy.

Frozen cells are preferably thawed quickly (e.g., in a water bathmaintained at 37-41° C.) and chilled immediately upon thawing.

It may be desirable to treat the cells in order to prevent cellularclumping upon thawing. To prevent clumping, various procedures can beused, including but not limited to the addition before and/or afterfreezing of Dnase (Spitzer et al., 1980, Cancer 45:3075-3085), lowmolecular weight dextran and citrate, hydroxyethyl starch (Stiff et al.,1983, Cryobiology 20:17-24), etc.

The cryoprotective agent, if toxic in humans, should be removed prior totherapeutic use of the thawed DC's.

One way in which to remove the cryoprotective agent is by dilution to aninsignificant concentration.

Once frozen DC's have been thawed and recovered, they are used toactivate T cells as described above with respect to non-frozen DC's.

As illustrated in the examples infra, in Section 8, extended life spanDC's have been cryopreserved, thawed and used to present antigen toactivate T cells in vitro.

5.2 Prostate Specific Antigens for Presentation by Dendritic Cells

A number of antigens or antigenic compositions are useful, according tothe present invention, for presentation by the DC's to activate T cellsfor prostate immunotherapeutics.

According to one embodiment, a lysate of LNCaP, a prostate cancer cellline, first described by Horoszewicz et al., 1980, Prog. Clin. Biol.Res. 37:115-132; 1983, Cancer Res. 43:1809-1818, is used as antigenpresented by the DC's. A crude cell lysate obtained simply by repetitivefreezing and thawing of LNCaP cells can be used as antigen. Anillustrative example of the use of this antigen is presented in Section6, infra. Alternatively, a membrane preparation of LNCaP cells, asdescribed by Horoszewicz et al., 1987, Anticancer Res. 7:927-936, can beused.

According to another embodiment, a prostate tumor cell lysate recoveredfrom surgical specimens can be used as antigen. For example, a sample ofa cancer patient's own tumor, obtained at biopsy or at surgicalresection, can be used to provide a cell lysate for antigen.Alternatively, a membrane preparation of tumor cells of a prostatecancer patient can be used as antigen.

According to another embodiment, purified prostate specific membraneantigen (PSMA, also known as CYPP antigen and as PSM antigen), whichspecifically reacts with monoclonal antibody 7E11-C.5 [(see generallyHoroszewicz et al., 1987, supra, U.S. Pat. No. 5,162,504; Feng et al.,1991, Proc. Am. Assoc. Cancer Res. 32:(Abs. 1418):238)] can be used asantigen. Cloning of the gene encoding the PSMA antigen has beendescribed by Israeli et al., 1994, Cancer Res. 54:1807-1811. Expressionof the cloned gene, e.g., in yeast cells, in a baculovirus expressionsystem or in a mammalian cell culture expression system, will provide aready source of the PSMA antigen for use according to the presentinvention.

According to another embodiment, an antigenic peptide having the aminoacid sequence LLHETDSAV (SEQ. ID. NO. 1) (designated “PSM-P1”) whichcorresponds to amino acid residues 4-12 PSMA can be used as antigen.According to another embodiment, an antigenic peptide having the aminoacid sequence ALFDIESKV (SEQ. ID. NO. 2) (designated “PSM-P2”) whichcorresponds to amino acid residues 711-719 of PSMA can be used asantigen.

According to another embodiment, an antigenic peptide having an aminoacid sequence XL(or M)XXXXXXV(or L) (SEQ. ID. NO. 3) (designated“PSM-X”) where X represents any amino acid residue can be used asantigen. This peptide resembles the HLA-A0201 binding motif, i.e., abinding motif of 9-10 amino acids with “anchor residues”, leucine andvaline found in HLA-A2+ patients. Grey et al., 1995, Cancer Surveys22:37-49. This peptide is preferably used as antigen for HLA-A2+patients. HLA-A2+ is expressed by a large proportion of patients. [See,Central Data Analysis Committee “Allele Frequencies”, Section 6.3,Tsuji, K. et al. (eds.), Tokyo University Press, pp. 1066-1077].

According to another embodiment, an antigenic peptide selected from thepeptides listed in Table 1A (below) can be used as antigen. The peptideslisted in Table 1A have amino acid sequences corresponding to fragmentsof PSM and have been matched to a binding motif of a specific haplotype.According to a preferred embodiment, the peptides are selected to bepresented by DC's to activate T cells of a patient which matched thehaplotype indicated for each peptide in Table 1A.

TABLE 1A PSM Peptides* Initial Amino Amino SEQ Acid Acid ID ID No.Haplotype Residue** Sequence NO. PSM-P3 A2  20 WLCAGALVL  4 PSM-P4 A2 27 VLAGGFFLL  5 PSM-P5 A2 109 ELAHYDVLL  6 PSM-P6 A2 260 NLNGAGDPL  7PSM-P7 A2 461 TLRVDCTPL  8 PSM-P8 A2 660 VLRMMNDQL  9 PSM-P9 A2 568PMFKYHLTV 10 PSM-P10 A2  57 NMKAFLDEL 11 PSM-P11 A2 469 LMYSLVHNL 12PSM-P12 A2 663 MMNDQLMFL 13 PSM-P16 A1 171 EGDLVYVNY 14 PSM-P17 A1 264AGDPLTPGY 15 PSM-P18 A1 463 RVDCTPLMY 16 PSM-P19 A1 143 LFEPPPPGY 17PSM-P20 A1 558 TYELVEKFY 18 PSM-P21 A1 701 AGESFPGIY 19 PSM-P22 A1 725WGEVKRQIY 20 PSM-P26 A11 398 IVRSFGTLKKE 21 PSM-P27 A11  63 DELKAENIKKF22 PSM-P28 A11 491 KSLYESWTKKS 23 PSM-P36 A24 448 AYINADSSI 24 PSM-P37A24 606 KYADKIYSI 25 PSM-P38 A24 298 GYYDAQKLL 26 PSM-P39 A24 624TYSVSFDSL 27 PSM-P40 A24 178 NYARTEDFF 28 PSM-P41 A24 227 LYSDPADYF 29PSM-P46 B3501 289 LPSIPVHPI 30 PSM-P47 B3501 501 SPSPEFSGM 31 *“PSMpeptides” refers to peptides having an amino acid sequence correspondingto a fragment of PSMA (a/k/a PSM). **“Initial Amino Acid Residue” refersto the residue number of the amino acid of PSM to which the first aminoacid of the peptide corresponds.

According to yet another embodiment, prostate specific antigen (PSA)(see Papsidero et al., 1980, Cancer Res. 40:2428-2432; McCormack et al.,1995, Urology 45(5):729-744) can be used as antigen.

According to another embodiment, an antigenic peptide selected from thepeptides listed in Table 1B (below) can be used as antigen. The peptideslisted in Table 1B have amino acid sequences corresponding to fragmentsby PSA and [PLEASE CONFIRM] have been matched to a binding motif of aspecific haplotype as indicated in Table 1B. According to a preferredembodiment, the peptide are presented by DC's to activate T cells ofpatients which match the haplotype indicated for each peptide in Table1B.

TABLE 1B PSA Peptides* Initial Amino Amino SEQ Iden. Acid Acid ID No.Haplotype Residue** Sequence NO. PSA-P1 A2  53 VLVHPQWVL 32 PSA-P2 A2171 KLQCVDLHV 33 PSA-P11 A1 235 ALPERPSLY 34 PSA-P21 A11  25 IVGGWECEK35 PSA-P22 A11 185 QVHPQKVTK 36 PSA-P23 A11 245 VVHYRKWIK 37 PSA-P31 A24152 CYASGWGSI 38 *“PSA peptides” refers to peptides having an amino acidsequence corresponding to a fragment of PSA. **“Initial Amino AcidResidue” refers to the residue number of the amino acid of PSA to whichthe first amino of the peptide corresponds.

According to still another embodiment, a prostate mucin antigen,recognized by monoclonal antibody PD41, described by Wright (U.S. Pat.No. 5,227,471 and No. 5,314,996; Beckett et al., 1991, Cancer Res.51:1326-1333) can be used as antigen. Alternatively, a crude lysate ofprostate tumor cells which bind to the antibody produced by thehybridoma cell line (ATCC HB 11094) and which express the PD41 mucinantigen could be used as antigen.

According to the present invention, DC's can be exposed to a desiredprostate cancer antigen or antigenic composition by incubating the DC'swith the antigen in in vitro culture medium. In one mode, the antigen,in aqueous soluble or aqueous suspension form, is added to cell culturemedium at the same time as the DC's and T cells to be stimulated areadded. See Section 6, infra, for an illustrative example of this method.As demonstrated in Section 6, the DC's advantageously took up antigenfor successful presentation to T cells. In another mode, antigens areintroduced to the cytosol of the DC's by alternate methods, includingbut not limited to osmotic lysis of pinocytic vesicles and the use of pHsensitive liposomes, etc. See, generally, Okada et al., 1982,“Introduction of Macromolecules Into Cultured mammalian Cells by OsmoticLysis of Pinocytic Vesicles”, Cell 29:33; Poste et al., 1976, “LipidVesicles as Carriers for Introducing Biologically Active Materials IntoCells”, Methods Cell Biol. 14:33; Reddy et al., 1991, “pH SensitiveLiposomes Provide an Efficient Means of Sensitizing Target Cells toClass I Restricted CTL Recognition of a Soluble Protein”, J. Immunol.Methods 141:157.

5.3 Applications or Methods of Use 5.3.1 Use of Dendritic Cells toPresent Prostate Antigen In Vitro

As mentioned above, according to one embodiment of the invention,isolated human DC's, exposed to a prostate specific antigen by any ofthe methods described above in Section 5.2, are used to activate T cellsin vitro against prostate cancer. The DC's can be used immediately afterexposure to antigen to stimulate T cells. Alternatively, the DC's can bemaintained in the presence of a combination of GM-CS and IL-4 prior tosimultaneous exposure to antigen and T cells.

T cells or a subset of T cells can be obtained from various lymphoidtissues for use as responder cells. Such tissues include but are notlimited to spleens, lymph nodes, and peripheral blood. The cells can beco-cultured with DC exposed to antigen as a mixed T cell population oras a purified T cell subset.

For example, it may be desired to culture purified CD8⁺ T cells withantigen exposed DC's to elicit prostate specific CTL. In addition, earlyelimination of CD4⁺ T cells may prevent the overgrowth of CD4⁺ cells ina mixed culture of both CD8⁺ and CD4⁺ T cells. T cell purification maybe achieved by positive, or negative selection, including but notlimited to, the use of antibodies directed to CD2, CD3, CD4, CD5, andCD8.

On the other hand, it may be desired to use a mixed population of CD4⁺and CD8⁺ T cells to elicit a prostate specific response encompassingboth a cytotoxic and T_(H) immune response.

According to a preferred embodiment, the T cells are obtained from thesame prostate cancer patient from which the DC's were obtained. Afterstimulation or activation in vitro, the autologous T cells areadministered to the patient to provoke and afford an immunoresponsewhich slows or inhibits prostate tumor growth.

For example, T cells are administered, by intravenous infusion, at dosesof about 10⁸-10⁹ cells/m² of body surface area (see, Ridell et al.,1992, Science 257: 238-241). Infusion can be repeated at desiredintervals, for example, monthly. Recipients are monitored during andafter T cell infusions for any evidence of adverse effects.

According to another embodiment, the T cells are obtained from aprostate cancer patient and the DC's which are used to stimulate thecells are obtained from an HLA-matched healthy donor. According to yetanother embodiment, both the T cells and the DC's are obtained from anHLA-matched healthy donor, e.g., a sibling of the prostate cancerpatient. This embodiment may be particularly advantageous, for example,when the patient is a late stage prostate cancer patient who has beentreated with radiation and/or chemotherapy agents and may not be able toprovide sufficient or efficient DC's. The T cells after stimulation, areadministered as described above.

5.3.2 Use of Dendritic Cells to Present Prostate Antigens In Vivo

According to another embodiment of the invention, DC's isolated from aprostate cancer patient are cultured, exposed in vitro to a prostatecancer antigen and after expansion and/or cryopreservation areadministered back to the patient to stimulate an immune response,including T cell activation, against the patient's cancer cells in vivo.Using this approach with the patient's own dendritic cells provides thefollowing advantages: (1) no foreign DNA is utilized; (2) infection ofcells for purposes of cDNA expression using various viral vectors areeliminated; (3) antigen is presented to dendritic cells in the form ofsoluble protein which will be taken into the dendritic cells andprocessed for MHC/peptide presentation of the cell surface; (4)dendritic cells express B7's on their surface alleviating the necessityto transfect this cDNA into dendritic cells; (5) the use of endogenousB7's on dendritic cell surface eliminates the need to provide T cellswith 11-2 or other cytokines either in the form of the cytokine itselfor transfection of the cDNA into specific cells; (6) all procedures arecarried out using the patient's own cells.

In practice, DC's obtained as described above in Section 5.1, areexposed in vitro to a prostate cancer antigen, washed and administeredto elicit an immune response or to augment an existing, albeit weak,response. As such, the DC's constitute an anti-prostate cancer vaccineand/or immunotherapeutic agent. DC's presenting a prostate specificantigen are administered, via intravenous infusion, at a dose of about10⁶-10⁸ cells. The immune response of the patient can be monitored.Infusion can be repeated at desired intervals based upon the patient'simmune response.

The following examples are presented for purposes of illustration onlyand are not intended to limit the scope of the invention in any way.

6. Example Use of Dendritic Cells to Stimulate Prostate Specific T Cells

The following example demonstrates that human dendritic cells, obtainedfrom prostate cancer patients, were able to elicit the proliferation ofboth helper and cytolytic T cells.

6.1. Materials and Methods 6.1.1. Cell Lines and Reagents

LNCaP, a prostate cancer cell line, Horoszewicz et al., 1983, LNCaPModel of Human Prostatic Carcinoma, Cancer Research, 43:1809-1818, (CRL1740, ATCC, Rockville, Md.), was maintained in culture in RPMI 1640.Granulocyte/macrophage colony stimulating factor (GM-CSF), recombinanthuman interleukin 2 (IL2) and interleukin 4 (IL4) were generous giftsfrom Amgen (Thousand Oaks, Calif.). Monoclonal antibodies Leu-6(anti-CD1a), Leu-4 (anti-CD3), Leu-3a (anti-CD4), Leu-2a (anti-CD8),Leu-M3 (anti-CD14), anti-HLA-DR (MHC Class II), and BB1 (anti-B7/BB1)were purchased from Becton-Dickinson, San Jose, Calif. Monoclonalantibodies S125-C1 (anti-CD19) and 3.9 (anti-CD11c) were purchased fromSigma, St. Louis, Mo.

6.1.2. Prostate Cancer Patients

Patients with a histologic confirmation of prostatic cancer wereselected for this study which included a signed informed consent. Fiftycc of heparinized peripheral blood were drawn every 2 weeks during theperiod of observation which continues. Details regarding clinical stage,hematologic status, and other relevant treatments are recorded in Table2. The American Urological System of staging was employed, i.e.,B₂=tumor confined to the prostate in both lobes, C₂=large locallyinvasive tumor, D₁=positive pelvic lymph node, D₂=metastatic disease.

TABLE 2 Clinical Profiles of Dendritic Cell Donors Average PBMC ClinicalBone Marrow Hematocrit Platelets yield# × DC Patient Age Stage HormonalStatus Status (Vol %) <100,000/mm³ 1000 yield* 1 64 D₁ HormoneRefractory, Intact 45 0 1000 8-12% Leupron + Flutamide 2 71 D₂ HormoneRefractory, Impaired 34 + 100 4-10% Post-Orchiectomy Ext. Radiation, 2 ×Sr⁸⁹ 3 68 C₂ Hormone Refractory, Ext. Radiation 46 0 1000 7-11%Post-Orchiectomy 4 65 D₂ Hormone Refractory, Ext. radiation 33 0 100010-14%  Post-Orchiectomy 5 75 B₂ Non-Treated Intact 47 0 1000 10-13%  670 D₂ Hormone Refractory, Impaired, 25 + 100  2-8% Post-Orchiectomy Ext.Radiation, Multiple Sites, 1 × Sr⁸⁹ 7 80 D₂ Hormone Refractory, Ext.Radiation, 29 + 1000 5-10% Post-Orchiectomy Multiple Sites 8 69 D₁Hormone Refractory, Intact 45 0 1000 8-11% Post-Orchiectomy R_(x)Flutamide 9 62 D₂ Hormone Refractory, Impaired, 33 0 300  6-9%Post-Orchiectomy Ext. Radiation, R_(x) Emcyt Multiple Sites, 1 × Sr⁸⁹ 1085 B₂ Intact Intact 46 0 900 9-13% #The number of PBMC isolated from 1ml of blood *The number of DC cultured for 7 days/the starting number ofPBMC × 100%. The average volume of blood drawn every session = 50 ml

Most of these patients are in clinical stages D₁ or D₂, hormonerefractory prostatic adenocarcinoma, and have undergone radiationtherapy. Seven patients have undergone orchiectomy, among whom threehave undergone Sr⁸⁹ therapy (patients 2, 6, and 9). Table 2 shows thatperipheral blood mononuclear cells (PBMC) yields from these threepatients were considerably lower (1-3×10⁵/cc) than those who were notgiven Sr⁸⁹ therapy (10⁶/cc).

6.1.3. Isolation of Dendritic Cells

Peripheral blood was drawn from prostate cancer patients and wassubjected to Lymphoprep (GIBCO-BRL, Gaithersburg, Md.) density gradientcentrifugation. The peripheral blood mononuclear cells (PBMC) isolatedwere plated in 24 well plates (10⁶-10⁷ cells/well) and were incubated ina humidified incubator (37° C., 5% Co₂) for 90 minutes.

Non-adherent cells were removed with the supernatant and the wells arewashed gently with warm (37° C.) OPTIMEM medium (GIBCO-BRL,Gaithersburg, Md.) and 5% FCS. Dendritic cell propagation medium (DCPM:OPTIMEM supplemented with 5% FCS, 500 units/ml GM-CSF and 500 units/mlIL-4) was added to the adherent cells (1 ml/well). These DC cells werecultured for 4-6 days before subculture 1:3 in DCPM.

The purity and identity of the isolated DC was confirmed by incubationwith monoclonal antibodies anti-CD1a, -CD3, -CD4, -CD8, -CD11c, -CD14,-CD19, —HLA-DR and -B7/BB1 for 30 minutes on ice followed by afluorescein-isothiocyanate labeled goat-anti-mouse Ig antibody for 30minutes on ice. Fluorescence binding was analyzed using a FACS SCAN flowcytometer (Becton Dickinson, San Jose, Calif.). In other words, thecultured DC cells were harvested by pipetting (leaving behind highlyadherent, bound macrophages) and were subjected to flow cytometeranalyses for surface expression of different protein markers for cellsof hematopoietic origin to confirm their DC identity. B7/BB1⁺, CD11c⁺,and HLA-DR⁺ (MHC class II) but CD14⁻, CD3⁻ and CD19⁻ confirms identityof the DC population.

6.1.4. T Cell Proliferation Assays

One million prostate cancer patients' PBMC were plated in microtiterplates in T cell media (TCM) consisting of RPMI 1640, HEPES,2-mercaptoethanol, L-glutamine and penicillin-streptomycin, supplementedwith 10% human AB serum (Sigma, St. Louis, Mo.) and 1 U/ml recombinanthuman Il-2. Ten thousand mitomycin-C inactivated autologous DC andantigen was added to the well prior to culture.

The antigens used in these assays were: (1) tetanus toxoid at 500 ng/ml(TT; Sigma, St. Louis, Mo.); (2) the lysate of LNCaP cells from anequivalent of 10⁵ LNCaP cells/ml; or (3) purified PSMA. Lysate wasprepared as described previously Topalian et al., 1994,Melanoma-Specific CD4+ T Lymphocytes Recognize Human Melanoma AntigensProcessed and Presented by Epstein-Barr Virus-Transformed B Cells, Int.J. Cancer 58:69-79. Briefly, 10⁷ LNCaP in 1 ml phosphate buffer saline(PBS) was subjected to cycles of repeated freezing in liquid nitrogenand quick thawing in a 37° C. water bath. Purified PSMA was prepared asfollows:

Preparation of the Protein-A 7E11-05 Column.

Protein-A agarose beads were washed with 10× volume 3 M NaCl pH=9.0. ThepH of the 2 ml of 7E11-05 monoclonal antibody solution was adjusted topH 9.0 and the NaCl concentration adjusted to 3 M. Antibody solution andbeads were mixed 1 hr at room temperature. After the incubation thebeads were washed with 10× volume 3 M NaCl, 50 mM Sodium Borate. Thebeads were resuspended in 10× volumes 3 M NaCl, 0.2 M Sodium Borate,pH=9.0. Dimethylpimeladate was added to a concentration to 20 mM. Themixture was allowed to mix 30 minutes at room temperature. The reactionwas stopped by washing the beads with 0.2 M methanolamine pH 8.0. Then0.2 M ethanolamine pH 8.0 was added at 10× volume and the mixture wasallowed to incubate with mixing for 2 hours. The final wash with 2×volume PBS 0.01% merthiolate.

Immunoprecipitation of PSMA From Semen.

Approximately 75 ml of human semen was collected from paid donors underthe WHO guidelines for fertility testing. The semen was spun down at10,000 RPMS for thirty minutes. The supernatant was then removed. Thepellet was washed two times with PBS pH=7.4 then subjected to 1 ml oflysis buffer [1% Triton X-100, 50 mM HEPES 10% glycerol, 15 mM MgCl₂, 1mM phenylmethylsulfonyl fluoride, and 1 mMethylenebis(oxyethylenenitrilo)tetraacetic acid for one hour. The lysatewas spun down at 10,000 RPMS for thirty minutes and the supernatant wascollected.

The 7E11-05 bound Protein-A beads were washed with 15 ml binding buffer(20 mM HEPES pH=7.5). After washing, the beads were added to 1 ml of theseminal pellet lysate and 2 ml of binding buffer. The mixture wasallowed to incubate over night.

The next day the beads were washed with 15 ml binding buffer, followedby 10 ml of wash buffer (10 mM Sodium Phosphate). The elution buffer(100 mM Glycine pH=1.8) was added at 2 ml volumes and the fractionscontaining PSMA were collected.

The cell suspension was then added to a T cell proliferation assays. Tcell cultures were incubated in a humidified 37° C. incubatorsupplemented with 5% CO₂ for 5 days prior to addition of 1 μCi³H-Thymidine/well. After a 24 h incubation, cells were harvested in asemi-automatic cell harvester (Skatron, Stevina, Va.) and radioactivityof the collected cells was determined. T cell proliferation was assessedby measurement of average ³HTdR incorporation.

6.2. Isolation and Characterization of Dendritic Cells from ProstateCancer Patients

DC were obtained from PBMC of prostate cancer patients as described inSection 6.1.3. above and cultured in DCPM as described. After 4-7 daysin culture, clusters of dividing cells started to form and became lessadherent to the tissue culture flask. These cells increased in size andshowed a typical dendritic morphology (results not shown). In additionto these slightly adherent cells, tightly adherent macrophages were alsopresent. The average number of cells with dendritic morphology obtainedafter a 7 day culture was 2−7×10⁶ from 50 ml peripheral blood,representing 4-14% of the starting number of PBMC cultured (see Table2).

In order to confirm the DC identity of the cultured cells, the cellswere harvested by pipetting (leaving tightly bound macrophages behind)and were subjected to flow cytometric analyses for surface expression ofdifferent protein markers for cells of hematopoietic origin. Results areillustrated in FIG. 1.

As illustrated in FIG. 1, the cultured cells do not express lineagespecific markers for T cells (CD3), B cells (CD19), or macrophages(CD14). CD1a, a marker for Langerhans cells (e.g., dendritic cellsisolated from the skin), was expressed at a high level early in theculture, but the level decreased when the cells were maintained inculture for more than 14 days. CD11c (beta-2-integrin) and HLA-DR wereexpressed at high levels while B7/BB1 was expressed at moderate levelsby these cells, confirming further the identify of the cultured cells asDC's.

These cultures ceased to expand after 2 passages, although the DC'smaintained their characteristic morphology and surface antigenexpression for up to one month when fed weekly with fresh dendritic cellpropagation medium (DCPM).

6.3. Stimulation of Antigen Specific T Cells by Dendritic Cells

In order to assess the capacity of the cultured DC's to present antigento and stimulate autologous T cells from the same patients, T cellproliferation assays were conducted as described above in Section 6.1.4.in triplicate. Tetanus toxoid (TT) was chosen as a representativeantigen in these experiments to determine whether patients' memory Tcells could be activated in vitro. Results are presented in FIG. 2.

FIG. 2 shows that autologous T cells cultured with the patients' DC'sand TT proliferated at levels significantly higher than backgroundlevels (in the absence DC) and at levels significantly higher than Tcells cultured with DC without TT, i.e., showing an autologous mixedlymphocyte reaction.

Thus, the results demonstrate that the presentation of TT by DC's isuseful for T cell proliferation. More particularly, the resultsdemonstrate that DC's obtained from prostate cancer patients are usefulto activate antigen specific T cell proliferation.

6.4. Stimulation of Prostate Specific T Cells by Dendritic Cells

The ability of DC's to present antigen specific for prostate cancer andstimulate autologous T cells of a prostate cancer patient wasdetermined. In one study, a crude cellular lysate of LNCaP cells, ametastatic prostate cancer cell line, was used as a representativeprostate cancer antigen in a T cell proliferation assay generally asdescribed above in Section 6.1.4. Results are illustrated in FIG. 3.

FIG. 3 shows that significant increases in ³HTdR incorporation wereobserved in 2 of 4 cases when both DC's and LNCaP lysates were includedin the T cell cultures. Thus, in 2/4 cases presentation of prostatespecific antigen stimulated autologous T cell proliferation in vitro. Itis our belief that the 2 negative cases with LNCaP lysate as antigenreflect the limitations of using a crude cellular lysate with variableconcentration of prostate antigen. Additional experiments using purifiedprostate-specific membrane antigen (PSMA) support this view (results notshown).

In another experiment, T cells proliferated as a result of DCpresentation of LNCaP lysate were expanded in culture, for 2 weeks, andsubjected to fluorescence flow cytometric analysis to determine therepresentation of the two T cell subtypes, i.e., cytolytic T lymphocytes(CTL) and helper T cells (T_(H)) elicited. In particular, T cellsproliferated as a result of DC presentation of LNCaP lysate wereexpanded, for two weeks, by culture in T cell propagation medium in thepresence of mitomycin C-inactivated autologous DC and LNCaP lysate(equivalent to 10⁴ LNCaP cell/ml). A 14 day old culture of T cells washarvested and analyzed for the expression of cytolytic T cell specificantigen; CD8, by fluorescence flow cytometry as described above. Resultsare illustrated in FIG. 4.

As demonstrated in FIG. 4, the CTL's (CD8+) represented about 40-50% ofthe T cells elicited against LNCaP by the DC presentation of LNCaPlysate. Thus, presentation of prostate antigen by autologous DC's isuseful to elicit both cytolic (CTL's) and helper (T_(H)) T cellsspecific for prostate cancer.

In yet another experiment, T cells (both CD4⁺ and CD8⁺) activated asdescribed above by DC's presenting LNCaP lysate as antigen demonstratedcytolytic activity using, as target cells, autologous DC's presentingLNCaP antigen.

Briefly, DC's were isolated from a prostate cancer patient, as describedin Section 6.2 and were exposed to LNCaP lysate as described above.Autologous T cells were obtained as PBMC's and cultured in the presenceof the LNCaP presenting DC's. T cells proliferated as a result ofprostate antigen presentation by the DC's were expanded for a total of21 days as described above.

The ability of the activated T cells to specifically lysis target cellswas assessed using the CytoTox 96™ assay (Promega, Madison; WI).Briefly, effector cells (the 21 day T cells including both CD4+ and CD8+cells) were incubated with 2×10⁴ target cells in T cell propagationmedium at effector: target cell ratios of 20:1, 10:1 and 3:1 for 5 hoursat 37° C. in a CO₂ incubator. Target cells included: autologous DC'salone or autologous DC's exposed to LNCaP lysate [equivalent of 10⁵cells/ml (DC+LNCaP Lysate)] overnight. Released lactate dehydrogenase, astable cytosolic enzyme that is released upon cell lysis was measuredfrom culture supernatants with a coupled enzymatic assay which wasmonitored by reading absorbance at 490 mm. All experiments wereperformed in triplicate. Results are illustrated in FIG. 5.

As shown in FIG. 5, T cells proliferated as a result of prostate antigenpresentation by DC's according to the present invention showed enhancedspecific lytic activity against cells presenting the same prostateantigen. Thus, presentation of prostate antigen by DC's is useful toelicit T cells having specific cytolytic activity against cellsexpressing prostate antigen.

7. Example Use of Cryopreserved Dendritic Cells to Stimulate ProstateSpecific T cells 7.1. Cryopreserved Dendritic Cells From a ProstateCancer Patient

Dendritic cells were isolated from PBMC of a prostate cancer patient asdescribed above in Section 6 and cultured, as described above, inSection 6, for 7 days in the presence of 500 units/ml GM-CSF and IL-4.

On day 7, the isolated DC's were harvested and cryopreserved using 90%fetal calf serum and 10% dimethylsulfoxide. The cryopreserved DC's werestored frozen for a period of time, thawed in a 37° C. water bath andtransferred to a 15 ml polypropylene tube and centrifuged at 1200 rpmfor 5 min. The thawed DC's were then resuspended in medium containing10% heat-inactivated human serum and counted.

One hundred thousand previously frozen T cells obtained from peripheralblood of the same prostate cancer patient (PBMC) were cultured in thepresence or absence of 10⁴ DC's together with 5 μl/well purified PSMA(see Section 6.1.4 above) in a total volume of 200 μl medium containing10% heat-inactivated human serum and 1 unit/ml IL-2. One μCi/well³H-Thymidine was added on day 6 and cultures were harvested 18 hourslater. ³H-TdR incorporation was counted in a liquid scintillationcounter. Each experiment was done in 4 replicates. Results are presentedin FIG. 6. The average cpm and standard deviation are shown in thegraph.

FIG. 6 demonstrates that a highly significant increase in ³HTdRincorporation was observed when both previously cryopreserved DC's andprostate specific antigen, i.e., PSMA, were included in the T cellcultures. Thus, presentation of prostate specific antigen by previouslycryopreserved DC's stimulated autologous T cell proliferation in vitro.The effect was significantly greater than that observed with antigenalone or with DC's alone but no exogenous prostate antigen.

7.2. Cryopreserved Dendritic Cells of Normal Patients

PBMC are obtained from a normal or healthy individual known not to besuffering from prostate cancer. The PBMC are cross-typed and therelevant HLA antigens expressed noted. Relevant HLA antigens includesuch as HLA-A, B, C and DR. DC are isolated from the PBMC's as describedin Section 6. The DC's are then cryopreserved as described in Section7.1 above.

The cryopreserved DC are used according to the present invention tostimulate T cells in vitro, from a similarly matched HLA-typedindividual or from a patient suffering from prostate cancer for use inthe cancer patient.

7.3. Alternate Protocol for Cryopreservation And Use of Dendritic Cells

Dendritic cells can be isolated from a prostate cancer patient or from ahealthy donor, cryopreserved and used according to the methods of theinvention as follows.

If desired, viable DC count, e.g., by trypan blue exclusion (Kucker,1977, Biochemical Methods in Cell Culture and Virology, Dwoder,Hutchinsen & Ross, Stroudsburg, Pa., pp. 18-19) and manual cell countingcan be performed.

The DC from the prostate cancer patient are cryopreserved using thefollowing protocol:

-   -   1. Gently resuspend cells to a concentration of 4×10⁶ viable        cells/ml, using a mixture of cold (4° C.) 50% autologous        plasma/RPMI-1640 or 50% heat-inactivated FCS/RPMI, and place the        suspension on ice.    -   2. In a cryovial containing 1 ml of a chilled sterile        cryoprotective medium of 20% DMSORPMI-1640, carefully layer a 1        ml portion of the above cell suspension on top of the        cryoprotective medium.    -   3. Approximately 10 minutes prior to freezing, slowly invert the        1:1 mixture to promote mixing, then place it on ice to allow        equilibrium between the cells and the cryoprotective medium.        NOTE: The “layered” tube should not remain unfrozen for very        long, so freezing should preferably be done within 20-30 minutes        after exposure of cells to DMSO/RPMI solution.    -   4. Place the vials in a freezing rack, which in turn is placed        in a 4° C. methanol bath, just deep enough to cover the cell        suspension. This is then placed in the bottom (to ensure proper        temperature) of a −80° C. freezer for at least 2 hours and less        than 24 hours.    -   5. After cells reach the frozen state, carefully and quickly        transfer them to a long term liquid nitrogen containment vessel.        A cryogenic storage vessel which can be used is the LR 1000        refrigerator (Union Carbide Corp., Indianapolis, Ind.) which        accommodates up to 40,000 cryules.

Following any desired length of time post-cryopreservation, the protocolbelow can be used to thaw the DC cells for use to stimulate autologous Tcells specific to prostate cancer according to the present invention.

-   -   1. Remove vial of frozen cells from liquid nitrogen. Immediately        the cell suspension by gently agitating the vial in a 37° C.        water bath until just a small amount of ice remains.    -   2. Aseptically, begin to add drop-wise, a chilled mixture of 50%        autologous serum/RPMI-1640 medium or 50% FCS/RPMI-1640 medium        with a slight mixing between each drop, until the suspension        volume is doubled.    -   3. Transfer this suspension to a larger centrifuge tube        (12-15 ml) and continue to add, drop-wise, 505 serum/RPMI        mixture with mixing between every other drop until the volume        reaches 6-7 ml. Diluent may now be added, drop-wise, with mixing        at every 0.5 ml increment until the volume reaches 9-10 ml.        (NOTE: The reason for stepwise addition of diluent is to prevent        osmotic shock to the cells as DMSO is diluted in the cell        suspension).    -   4. Pellet cells by centrifugation at 4° C. 200×g, for 10        minutes. Aspirate the supernatant.    -   5. Slowly add drop-wise 1 ml of chilled 20% autologous        serum/RPMI-1640 mixture to the pellet. “Resuspend” the pellet by        gently “flicking” the tube with a finger. After the pellet is        resuspended (clumps may remain), resuspend it further by gently        aspirating up and down with a 1 ml pipet.    -   6. Add an additional 4 ml chilled 20% autologous serum/RPMI,        dropwise, with mixing between every drop; then add 0.5 ml as        volume increases, as previously described.    -   7. Pellet cells by centrifugation at 4° C. 200×g, for 10        minutes. Aspirate the supernatant.    -   8. Resuspend with 2-5 ml of chilled 20% serum/RPMI mixture.    -   9. Perform cell counts (e.g., by use of a hemocytometer) and        viability testing (e.g., by trypan blue exclusion).

Loss of cells due to clumping during the stepwise removal of DMSO can bediminished by including Dnase (20 U per 2×10⁶ cells) or low molecularweight dextran and citrate (to reduce the Ph to 6.5).

8. Example Use of Extended Life Span Dendritic Cells to StimulateProstate Specific T Cells

Extended life span dendritic cells were prepared according to thepresent invention as follows:

DC's were isolated from human peripheral blood and cultured for 4-6 daysas described above in Section 6.1.3. After 4-6 days in culture, in thepresence of GM-CSF and IL-2, the DC's were infected with Epstein BarrVirus (EBV) generally as described by Walls & Crawford, in Lymphocytes:A Practical Approach, GCB Klaus, ed., IRL Press, Oxford, England, pp.149-162. Briefly, DC's were harvested (˜3−5×10⁶ cells) and resuspendedin a 5 ml B95-8 (an EBV-producing marmoset cell line, American TypeCulture Collection, Rockville, Md.) culture supernatant. The cellsuspension was transferred to a 25 cm² flask (Nunc, Inc., Naperville,Ill.) and incubated in a 37° C. CO₂ incubator for 24 hours. Five ml offresh DCPM were added to the culture. Clusters of growing cells wereobserved 2-3 weeks post-infection. Extended life span DC cells weresubcultured weekly, at 3-5 weeks post-infection.

The ability of the extended life span DC's to present antigen tostimulate autologous T cells of a prostate cancer patient wasdetermined. In this study, either tetanus toxoid (TT) at 500 ng/ml or alysate of LNCaP cells at an equivalent of 10⁴ cells/ml was used asrepresentative antigen.

The autologous T cells used in the proliferation assay were obtainedfrom peripheral blood and had been previously cryopreserved. One hundredthousand thawed T cells (designated “PBMC”) were cultured in completemedium containing 10% heat-inactivated human serum and 1 unit/ml IL-2 asfollows: in the presence of LNCaP antigen alone (+LNCaP); in thepresence of TT antigen alone (+TT); in the presence of EBV-transformedDC's (+EBV cells); in the presence of EBV-transformed dendritic cellsplus LNCaP antigen (+EBV-cells+LNCaP); or in the presence ofEBV-transformed dendritic cells plus TT (+EBV-cells+TT). T cells in thepresence of medium alone (PBMC+medium only) served as a backgroundsample.

One μCi/well ³H-Thymidine (³H-TdR) was added to all wells on day 6 andcultures were harvested 18 hours later. ³H-TdR incorporation wasassessed using a liquid scintillation counter. Each experiment wasconducted in triplicate. ³H-TdR incorporation, average cpm for eachculture, was determined after subtracting average background cpm.Results are presented in FIG. 7.

FIG. 7 demonstrates that extended life span human dendritic cells,exposed either to TT antigen or PSMA antigen, significantly increased³HTdR incorporation in previously cryopreserved T cell cultures. Thus,it is clear that extended life span DC's are useful to present prostatespecific antigen to stimulate autologous T cell proliferation in vitro.

9. Example Use of a PSMA Peptide to Stimulate T Cells

The ability of a peptide having the amino acid sequence, LLHETDSAV (SEQ.ID. NO. 1) (designated PSM-P1), which corresponds to amino acid residues4-12 of the PSMA antigen as deduced from cDNA (of the PSMA gene) tostimulate T cells was assessed in order to determine whetherpresentation of such antigenic peptide would be useful using the methodsof the present invention. The following data show that this antigenicpeptide is suitable for use in the methods of the invention.

A peptide having amino acid sequence LLHETDSAV (designated PSM-P1peptide) was synthesized and purified by Genemed Biotechnologies (SanFrancisco, Calif.).

One hundred thousand previously frozen T cells (designated “PBMC”)obtained from a prostate cancer patient were cultured in the presence orabsence of 20

PSMA peptide in complete medium containing 10% heat-inactivated humanserum and 1 unit/ml IL2. One μCi/well ³H-Thymidine was added on day 6and cultures were harvested 18 hours later. ³H-TdR incorporation wascounted in a liquid scintillation counter. Each experiment was done in 4replicates. The average cpm and standard deviation are shown in FIG. 8.

As illustrated in FIG. 8, the antigenic peptide corresponding to aminoacid residues 4-12 of PSMA peptide elicited a greatly enhanced T cellproliferation compared to T cells in the absence of this peptide.

The T cells proliferated as a result of the PSMA peptide were expandedin culture for 5 days, in complete medium containing 2 μM PSMA peptideand 1 unit/ml Il2 for 5 days prior to harvest. One million cells wereincubated for 30 minutes on ice with anti-CD4, anti-CD8 antibodies ormedium alone, followed by another 30 minute incubation with fluoresceinisothiocyanate labelled-2° antibody. Fluorescence-bound was analyzed ina FACScan (Becton Dickinson, San Jose, Calif.) flow cytometer asdescribed above. The percent CD4+ and CD8+ cells were calculated.Results are illustrated in FIG. 9.

As shown in FIG. 9, the CTL's represented about 41% of the T cellsstimulated by the PSMA peptide antigen. Thus, this peptide antigenshould be useful to be presented by DC's to T cells to elicit prostatespecific responses, both cytolytic (CTL's) and helper (T_(H)) cellsagainst prostate cancer according to the methods of the invention.

In another series of experiments, the ability of a peptide, having theamino acid sequence, ALFDIESKV (SEQ. ID. NO. 2) (designated PSM-P2), tostimulate T cells was assessed. The following data show that thisantigenic peptide is suitable for use in the methods of the invention.

Peptide PSM-P2 was synthesized and purified.

One hundred thousand T cells, i.e. PBMC obtained from HLA-A2(+) orHLA-A2(−) prostate cancer patients were cultured in the presence orabsence (i.e. “medium control”) of 20 μg PSM-P2 peptide for five (5)days in a humidified, 37° C., 5% CO₂ incubator. On day 6, 1 μCi³H-Thymidine was added to each well. After another 24 h incubation,cells were harvested and the ³H-TdR incorporated was counted in a liquidscintillation counter. Each experiment was done in triplicate. Resultsare presented in FIG. 10 as average cpm and standard error of the mean.

As demonstrated in FIG. 10, the peptide corresponding to amino acidresidues 711-719 of PSMA elicited a greatly enhanced cell proliferationof HLA-A2(T cells compared to HLA-A2(+) T cells in the absence of thispeptide. A less dramatic enhanced T cell proliferation was observed whenHLA-A2 (−) T cells were exposed to this peptide.

10. Example Use of Dendritic Cells to Produce an Immune Response inHuman Prostate Cancer Patients

A phase 1 clinical trial has been established using five treatmentgroups of late stage prostate cancer patients. The groups were treatedas follows: Group I, received peptide PSM-P1 having amino acid sequenceLLHETDSAV (SEQ. ID. NO 1) only at 0.2, 2.0 and 20.0 μg; Group II,peptide PSM-P2 having amino acid sequence ALFDIESKV (SEQ. ID. NO. 2)only at 0.2, 2.0 and 20.0 μg; Group III, 1×10⁶ (#3-4) or 5×10⁶ (#1, 2)autologous dendritic cells only; Group IV, up to 2×10⁷ autologousdendritic cells loaded with PSM-P1 (SEQ. ID. NO. 1); and Group V, up to2×10⁷ autologous DC's loaded with PSM-P2 (SEQ. ID. NO. 2). Alltreatments were delivered by infusion. Patients were monitored 7 dayspost-infusion and their immune function was analyzed by assessing T cellproliferation pre- and post-infusion using ³H-TdR incorporation asdescribed above in Section 9. Results, after the first infusion ofGroups I-V are presented in FIG. 11 (A-E).

As shown in FIG. 11A, patients receiving peptide PSM-P1 alone did notdemonstrate any increase in T cell proliferation, regardless of theirHLA-type. As shown in FIG. 11B, only one patient (out of 8 patients)receiving peptide PSM-P2 alone demonstrated an increased T cellproliferation. The sole positive patient was an HLA-A2(+) patient.

As demonstrated in FIG. 11C, patients receiving dendritic cells only didnot demonstrate any increase in T cell proliferation. As indicated inFIG. 11C, three of the four patients were HLA-2(A)(+) and one wasHLA-2(A)(−).

In complete contrast to the results presented in FIG. 11 (A-C), resultsin FIGS. 11(D-E) demonstrate that administration of DC's exposed to andpresenting either peptide PSM-P1 or PSM-P2 induced an enhanced T cellproliferation. More particularly, patients receiving dendritic cellscontaining PSM-P1 peptide showed an increased T cell proliferation in 3of 5 HLA-A(2)(+) as illustrated in FIG. 11(D). In addition, the soleHLA-A(2)(−) patient did not demonstrate an increased T cellproliferation. These results demonstrate firstly, that administration ofdendritic cells in Combination with the PSM-P1 peptide effectivelyproduced an immune response in late stage prostate cancer patients.Secondly, it appears that the immune response is an HLA-A(2) restrictedeffect when this peptide was used. The two patients receiving dendriticcells containing PSM-P2 peptide also showed an increased T cellproliferation as illustrated in FIG. 11(E). Both of these patients wereHLA-A2(+).

Based on the results obtained, we have concluded that dendritic cellsexposed either to PSM-P1 or to PSM-P2 peptide are useful to produce animmune response in prostate cancer patients and that the methods andcompositions of this invention are advantageously useful for cancerimmunotherapy.

The present invention is not to be limited in scope by the exemplifiedembodiments which are intended as illustrations of single aspects of theinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications are intended to fall within the scope of the appendedclaims.

All publications cited herein are incorporated by reference in theirentirety.

1. A composition comprising an isolated cell population having humandendritic cells, wherein said cell population has been cultured in thepresence of granulocyte-macrophage colony stimulating factor (GM-CSF),interleukin 4 (IL-4), and exposed in vitro to a soluble prostateantigen, the cell population having an increased ability to activate Tcells specific to the prostate antigen as compared to a similar isolatedcell population cultured in the presence of granulocyte-macrophagecolony stimulating factor (GM-CSF), and interleukin 4 (IL-4) that hasnot been exposed in vitro to the prostate antigen.
 2. The compositionaccording to claim 1, in which the prostate antigen is a lysate of LNCaPcells, a membrane preparation of LNCaP cells, a lysate of prostate tumorcells from a prostate cancer patient, a membrane preparation of prostatetumor cells from a prostate cancer patient, isolated prostate specificmembrane antigen (PSMA), purified prostate specific membrane antigen(PSMA), a peptide having the amino acid sequence LLHETDSAV (SEQ ID NO:1), a peptide having the amino acid sequence ALFDIESKV (SEQ ID NO: 2), apeptide having the amino acid sequence XL(or M)XXXXXV(or L) (SEQ ID NO:3), where X represents any amino acid, purified prostate specificantigen (PSA), or a purified prostate mucin antigen recognized bymonoclonal antibody PD41.
 3. The composition according to claim 1, inwhich the prostate antigen is selected from the group consisting of:WLCAGALVL; (SEQ. ID. NO. 4) VLAGGFFLL; (SEQ. ID. NO. 5) ELAHYDVIA;(SEQ. ID. NO. 6) NLNGAGDPL; (SEQ. ID. NO. 7) TLRVDCTPL; (SEQ. ID. NO. 8)VLRMMNDQL; (SEQ. ID. NO. 9) PMFKYHLTV; (SEQ. ID. NO. 10) NMKAFLDEL;(SEQ. ID. NO. 11) LMYSLVHNL; (SEQ. ID. NO. 12) MMNDQLMFL;(SEQ. ID. NO. 13) EGDLVYVNY; (SEQ. ID. NO. 14) AGDPLTPGY;(SEQ. ID. NO. 15) RVDCTPLMY; (SEQ. ID. NO. 16) LFEPPPPGY;(SEQ. ID. NO. 17) TYELVEKFY; (SEQ. ID. NO. 18) AGESFPGIY;(SEQ. ID. NO. 19) WGEVKRQIY; (SEQ. ID. NO. 20) IVRSFGTLKKE;(SEQ. ID. NO. 21) DELKAENIKKF; (SEQ. ID. NO. 22) KSLYESWTKKS;(SEQ. ID. NO. 23) AYINADSSI; (SEQ. ID. NO. 24) KYADKIYSI;(SEQ. ID. NO. 25) GYYDAQKLL; (SEQ. ID. NO. 26) TYSVSFDSL;(SEQ. ID. NO. 27) NYARTEDFF; (SEQ. ID. NO. 28) LYSDPADYF;(SEQ. ID. NO. 29) LPSIPVHPI; (SEQ. ID. NO. 30) SPSPEFSGM;(SEQ. ID. NO. 31) VLVHPQWUL; (SEQ. ID. NO. 32) KLQCVDLHV;(SEQ. ID. NO. 33) ALPERPSLY; (SEQ. ID. NO. 34) JVGGWECEK;(SEQ. ID. NO. 35) QVHPQKVTK; (SEQ. ID. NO. 36) VVHYRKWIK;(SEQ. ID. NO. 37) CYASGWGSI. (SEQ. ID. NO. 38)


4. The composition according to claim 1, in which the dendritic cellsare extended life span dendritic cells.
 5. The composition according toclaim 1, in which the prostate tissue antigen is: WLCAGALVL;(SEQ. ID. NO. 4) VLAGGFFLL; (SEQ. ID. NO. 5) ELAHYDVLL; (SEQ. ID. NO. 6)NLNGAGDPL; (SEQ. ID. NO. 7) TLRVDCTPL; (SEQ. ID. NO. 8) VLRMMNDQL;(SEQ. ID. NO. 9) PMFKYHLTV; (SEQ. ID. NO. 10) NMKAFLDEL;(SEQ. ID. NO. 11) LMYSLVHNL; (SEQ. ID. NO. 12) MMNDQLMFL;(SEQ. ID. NO. 13) EGDLVYVNY; (SEQ. ID. NO. 14) AGDPLTPGY;(SEQ. ID. NO. 15) RVDCTPLMY; (SEQ. ID. NO. 16) LFEPPPPGY;(SEQ. ID. NO. 17) TYELVEKFY; (SEQ. ID. NO. 18) AGESFPGIY;(SEQ. ID. NO. 19) WGEVKRQIY; (SEQ. ID. NO. 20) IVRSFGTLKKE;(SEQ. ID. NO. 21) DELKAENIKKF; (SEQ. ID. NO. 22) KSLYESWTKKS;(SEQ. ID. NO. 23) AYINADSSI; (SEQ. ID. NO. 24) KYADKIYSI;(SEQ. ID. NO. 25) GYYDAQKLL; (SEQ. ID. NO. 26) TYSVSFDSL;(SEQ. ID. NO. 27) NYARTEDFF; (SEQ. ID. NO. 28) LYSDPADYF;(SEQ. ID. NO. 29) LPSIPVHPI; (SEQ. ID. NO. 30) SPSPEFSGM;(SEQ. ID, NO. 31) VLVHPQWUL; (SEQ. ID. NO. 32) KLQCVDLHV;(SEQ. ID. NO. 33) ALPERPSLY; (SEQ. ID. NO. 34) JVGGWECEK;(SEQ. ID. NO. 35) QVHPQKVTK; (SEQ. ID. NO. 36) VVHYRKWIK;(SEQ. ID. NO. 37) or CYASGWGSI. (SEQ. ID. NO. 38)


6. The composition according to claim 1, in which the dendritic cellshave been cryopreserved prior to exposure in vitro to the prostateantigen, wherein said dendritic cells retain the ability to take up andpresent antigen.
 7. The composition according to claim 6, in which theprostate antigen is a lysate of LNCaP cells, a membrane preparation ofLNCaP cells, a lysate of prostate tumor cells from a prostate cancerpatient, a membrane preparation of prostate tumor cells from a prostatecancer patient, isolated prostate specific membrane antigen (PSMA),purified prostate specific membrane antigen (PSMA), a peptide having theamino acid sequence LLHETDSAV (SEQ. ID. NO. 1), a peptide having theamino acid sequence ALFDIESKV (SEQ. ID. NO. 2), a peptide having theamino acid sequence XL(or M)XXXXXV(or L) (SEQ. ID. NO. 3), where Xrepresents any amino acid, purified prostate specific antigen (PSA), ora purified prostate mucin antigen recognized by monoclonal antibodyPD41.
 8. The composition according to claim 6, in which the dendriticcells are extended life dendritic cells.
 9. The composition according toclaim 1, wherein said dendritic cells can activate 2 to 3 fold more Tcells specific to the prostate antigen as compared to an isolated cellpopulation cultured in the presence of granulocyte-macrophage colonystimulating factor (GM-CSF) and interleukin 4 (IL-4) that has not beenexposed in vitro to the prostate antigen.
 10. The composition accordingto claim 1, wherein the human dendritic cells are immature dendriticcells.
 11. The composition according to claim 1, wherein the T cells areCD4⁺.
 12. The composition according to claim 1, wherein the T cells areCD8⁺.
 13. The composition according to claim 1, wherein the dendriticcells are isolated from a prostate cancer patient.
 14. The compositionaccording to claim 1, wherein the dendritic cells are isolated from anormal individual.
 15. The composition according to claim 14, whereinthe dendritic cells are HLA-matched for a recipient.